DESCRIPTION (applicant's abstract): In this R21 (exploratory/developmental) grant application, we wish to address a central problem concerning the assembly of the phospholipid bilayer of biological membranes, i.e., what is the mechanism by which phospholipids are translocated across biogenic (self-synthesizing) membranes? This is an underappreciated, understudied area, and we believe that despite the intrinsic experimental difficulties in measuring phospholipid flip-flop, we are in an unique position to approach this longstanding problem. Newly synthesized phospholipids are located in the cytoplasmic leaflet of biogenic membranes such as the endoplasmic reticulum (ER) of eukaryotic cells and the cytoplasmic membrane (bCM) of bacteria. While these lipids can diffuse laterally in the membrane leaflet in which they are situated, transverse diffusion or flip-flop is thermodynamically restricted and does not occur spontaneously. However, such movement is essential to propagate a membrane bilayer, and a number of reports indicate that phospholipids translocate rapidly across the ER and bCM. These reports also indicate that translocation is bidirectional and occurs by a facilitated diffusion process requiring no metabolic energy input. This observation rules out the participation of the ABC family of transporters which are involved in metabolic energy-dependent, vectorial transport of solutes and some lipids. Thus the molecular mechanism by which phospholipids are translocated across the ER and bCM is unknown. We hypothesize that there exists a novel class of lipid translocators that facilitate diffusion of phospholipids in a metabolic energy-independent fashion across the ER and bCM bilayers. We term these translocators biogenic membrane flippases, to distinguish them from metabolic energy-requiring lipid translocators (like the ABC transporters) that are driven by ATP hydrolysis or protonmotive force. Our aim is to identify a biogenic membrane flippase by biochemical and genetic means, thus providing a direct test of our hypothesis. To do this we will explore biochemical reconstitution/protein purification approaches using Bacillus membranes as a starting point, as well as forward and reverse genetic strategies utilizing Escherichia coli. The last of these involves a comparative genomics approach, making novel use of available sequenced genome databases. Our long term goal is to obtain a molecular definition of the mechanism of phospholipid flip-flop. We expect that our analyses will not only impact current understanding of membrane biogenesis, but also contribute to an understanding of glycolipid flip-flop events that are essential in the assembly of cell surface components.